Proteases play essential roles in many disease processes such as Alzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compounds that block or enhance their activity have potential as therapeutic agents. Because the normal substrates of peptidases are linear peptides and because established procedures exist for making non-peptidic analogs, compounds that effect the activity of proteases are natural subjects of combinatorial chemistry. Accordingly, screening compounds produced by combinatorial chemistry requires a convenient enzymatic assays.
Apoptosis is a physiological mechanism of cell death which involves the fragmentation of a cell into membrane-bound particles. The process of apoptosis is involved in a variety of normal and pathogenic biological events, both during development and in adulthood. Agents which affect apoptosis may have therapeutic utility in treating diseases and disorders characterized by aberrant cell proliferation or death (reviewed in Hoeppner et al., Biochim. Biophys. Acta 242: 217-220, 1966; Thompson, Science 267:1456-1462, 1995). Techniques for detection of apoptosis may be useful to screen for potential therapeutic agents that may induce or prevent apoptosis.
Caspases are a class of proteins central to the apoptotic program and are cysteine protease having specificity for aspartate as a substrate cleavage site. These proteases are primarily responsible for the degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. For example, one of the caspases identified in humans was previously known as the interleukin-1β (IL-1β) converting enzyme (ICE), a cysteine protease responsible for the processing of pro-IL-1β to the active cytokine. Overexpression of ICE in Rat-1 fibroblasts induces apoptosis (Miura et al., Cell 75:653, 1993).
The Caspase family proteases have been found to play an essential role in the intracellular pathway of apoptosis (reviewed in Martin et al., Cell 82:349-352 1995). ICE itself is not a mediator of apoptosis in most mammalian cell types. Rather, a family of homologous proteases comprising at least nine human ICE family proteases have been identified to date (ICE, CPP32/apopain/Yama, ICH-1, TX/ICH-2/ICErel III, ICErel III, MI-L1/MH-3/ICE-LAP3, Mch2, FLICE/Mch5, ICE-LAP6/Mch6), each of which leads to apoptosis when over-expressed in a proteolytically active form in cultured mammalian cells (Miura et al., Cell 75:653-660 1993; Wang et al., Cell 78:739-750 1994; Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-30764 1994; Faucheu et al., EMBO J. 14:1914-22, 1995; Kamens et al., J. Biol. Chem, 270:15250-15256, 1995; Alnenui et al., J. Biol. Chem. 270:4312-4317, 1995; Fernandes-Alnemri et al., Cancer Res. 55:6045-6052, 1995; Lippke et al., J. Biol. Chem. 271:1825-1828, 1996; Muzio et al., Cell 85:817-827, 1996; Duan et al., J. Biol. Chem. 271:16720-16724, 1996). Moreover, treatment of cells with apoptotic stimuli increases ICE-like proteolytic activity in cell extracts (Los et al., Nature 375:81-83, 1995; Enari et al., Nature 380:723-726, 1996).
Degradation of specific cellular proteins following the activation of an ICE-like protease, has also been associated with apoptosis. For example, poly(ADP-ribose)polymerase (PARP) is cleaved specifically during apoptosis in mammalian cells (Kaufmann et al., Cancer Res, 53:3976-3985, 1993) and is an excellent substrate in vitro for several ICE homologues (Tewari et al., Cell 81:801-809, 1995; Nicholson et al., Nature 375:37-43, 1995; Gu et al., J. Biol. Chem. 270:18715-18718, 1995; Fernandes-Alnemri et al., Cancer Res. 55:2737-2742, 1995, Fernandes-Alnemri et al., ibid.; Lippke et al., J. Biol. Chem. 271:1825-1828, 1996). Protease inhibitors which block the activity of ICE homologues prevent not only apoptosis, but PARP degradation as well (Schlegel et al., ibid.).
Due to the inadequacies in many of the known methods for the detection of cell apoptosis, there continues to be a need for new, selective methods of detection.